AUTOMATIC AND QUANTITATIVE ANALYSIS OF NGF RECEPTORS CO-ENDOCYTOSIS

VILLALTA JI, IACARUSO MF, JARES-ERIJMAN EA, GALLI S, PIETRASANTA LI

Hotel Casa Serrana, Huerta Grande, Cordoba,

Congreso; I reunión conjunta neurociencias (IRCN),; 2009

Spatial colocalization analysis of two fluorescently labeled molecules in optical microscopy is of major importance in cell biology. Qualitative colocalization analysis is generally visual-based and therefore highly prone to random error and bias. New methods to quantitatively analyze colocalization through the evaluation of overlapping pixels, based on global statistic analysis of pixel intensity distributions have been recently developed (Manders et al., 2003; Costes et al., 2004; Li et al., 2004). Still, the majority of colocalization situations demand customized approaches. We developed an algorithm that quantitatively and automatically tracks colocalization without the bias of visual interpretation and simultaneously overcomes definite inconvenients not contemplated by former algorithms: i) it detects colocalization in images where fluorophore distribution is mainly anticolocalizing and thus are negative correlated; ii) it distinguishes colocalizing pixels in images with high density of fluorescent molecules; iii) it can be applied to the whole images or to a definite region with the same level of accuracy. In addition, we redefined the utility of the colocalization coefficients by constructing informative maps of the distribution of these coefficients throughout the image rather than obtaining single less informative values for the whole image. In this way we could directly observe the regions inside the image where colocalization is relevant. We integrated these analyses into a simple user-friendly graphical user interface (GUI) for MATLAB designed for easy accessibility. With this new method we determined the characteristic of the endocytosis of the NGF receptors TrkA and p75 when co-expressed in PC12 cells. We observed that upon overexpression of TrkA-CFP and p75-YFP three populations of endosomes arose: those containing only p75, only TrkA or both receptors. This finding may shed light on the mechanism that underlies the cooperation of these receptors on NGF signal transduction.